Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua L, extracts, which has been used to treat malaria and a variety of other ailments for nearly 2000 years. Artemisinin and its derivatives, such as dihydroartemisinin, artemether, artesunate, arteether, propylcarbonate dihydroartemisinin, and artelinic acid, are most commonly known as potent anti-malarial agents. The artemisinin molecule contains an endoperoxide moiety, or oxygen bridge. The anti-malarial activity of artemisinin is due to the reaction between its endoperoxide bridge and intra-parasitic heme that generates free radicals, causing cell death.
The artemisinin molecule and related compounds have been studied extensively covering aspects such as characterization, total synthesis, and understanding of the mechanism of action through QSAR studies. These studies have unveiled a large amount of information about the artemisinin and related endoperoxide compounds and have resulted in a large number of published and patented literatures (See, Bez, G., et al., Current Organic Chemistry 7:1231-1255, 2003). For example, the endoperoxide function has been shown to be essential for the antimalarial activity of artemisinin (Gu, Acta Pharmacol. Sinica 1(1):48-50, 1980 Abstract). The total synthesis of (+)-artemisinin has been reported (Avery, M. A., et al., Tetrahedron Letters 28:4629-4632, 1987). The same group also synthesized several simplified analogues of artemisinin (Avery, 1987). Lin et al. reported a new series of hydrolytically stable and water-soluble dihydroartemisinin derivatives with optically active side chains as potential antimalarial agents (Lin, 1989). Imakura et al. reported the study of acid degradation products of artemisinin and their structure-activity relationships (Imakura, Y., et al., Heterocycles 31(6):1011-1016, Jun. 1, 1990. Abstract). Zaman et al. reported the aspects of the chemistry and biological activity of artemisinin and related antimalarials. (Zaman, S. S., and R. P. Sharma, Heterocycles 32:1593-1638, 1991). Peters et al. evaluated the activities of some synthetic artemisinin endoperoxide 1,2,4-trioxanes against several lines of Plasmodium berghei and P. yoelii ssp. in vivo (Peters, W., et al., Ann. Trop. Med. Parasit. 87(1):9-16, 1993). The results from these studies have enabled scientists during the 1990s to delineate the basic structural requirement for artemisinin-related endoperoxides—the 1,2,4-trioxane ring system—as the essential pharmacophore for artemisinin. Since then, interest in artemisinin has persisted. Benoit-Vical et al. reported the in vitro and in vivo potentiation of artemisinin and synthetic endoperoxide antimalarial drugs in 2000 (Benoit-Vical, F., et al., Antimicrobial Agents and Chemotherapy 44(10):2836-2841, 2000). Recently, Anfosso et al. used microarray expression profiles of angiogenesis-related genes to predict tumor cell response to artemisinin (Anfosso, L. et al., Pharmacogenomics Journal, 2006, pp. 1-10).
As a result of an apparent association between the endoperoxide functional group and antimalarial activity of artemisinin, a substantial effort has been devoted to developing new peroxide antimalarials (Vennerstrom, J. L., and J. W. Eaton, Journal of Medicinal Chemistry 31(7):1269-1277, 1988). Motivated by the structure and pharmacological mechanism of artemisinin, a large number of molecules containing the core pharmacophore, 1,2,4-trioxane, as well as its close analogue, 1,2,4,5-tetraoxane, and other endoperoxides have been synthesized and studied (U.S. Pat. No. 6,906,205, U.S. Pat. No. 6,486,199).
1,2,4-Trioxane itself has not been isolated or characterized. The tremendous amount of literature in the field suggests that it is the discovery of artemisinin with its novel 1,2,4-trioxane heterocyclic pharmacophore that initiated the development of 1,2,4-trioxanes, 1,2,4,5-tetraoxanes, and other artemisinin-related endoperoxides derivatives. Rational design of structurally simpler analogs of artemisinin has led to the synthesis of various racemic 1,2,4-trioxanes displaying potent antimalarial activities (U.S. Pat. No. 5,225,437). One group reported the development of dispiro-1,2,4,5-tetraoxanes as endoperoxide antimalarial drugs (Vennerstrom, J. L., et al., Journal of Medicinal Chemistry 35:3023-3027, 1992), as well as identification of a series of 1,2,4-trioxolane antimalarial drug candidates (US 2005/0256185).
Cancer cells have a significantly higher influx of iron than normal cells. Accordingly, it has been shown that artemisinin and artemisinin analogs are cytotoxic against established tumors and tumor cell lines (see, e.g., Woerdenbag, et al. (1993) J. Nat. Prod. 56(6):849-56; Lai and Singh (1995) Cancer Lett. 91:41-6; Efferth, et al. (2001) Int. J. Oncol. 18:767-73; Li, et al. (2001) Bioorg. Med. Chem. Lett. 11:5-8; Singh and Lai (2001) Life Sci. 70:49-56; Efferth, et al. (2002) Biochem. Pharmacol. 64:617-23; Efferth, et al. (2002) Blood Cells, Molecules and Diseases 28(2): 160-8; Sadava, et al. (2002) Cancer Lett. 179: 151-6; Singh and Lai (2004) Anticancer Res. 24(4):2277-80; Lai, et al. (2005) Expert Opin Ther Targets. 9(5):995-1007; Lai and Singh (2006) Cancer Lett. 231(1):43-8).
Similarly, artemisinin and its derivatives are also selectively cytotoxic to other cells with uncontrolled elevated free iron levels. Representative cells with elevated free iron level include cancer cells, pathogenic organisms, and abnormally hyperproliferating cells found in conditions, such as restenosis, arthritis, hyperplasia, and psoriasis (see, e.g., Golenser, et al. (2006) Int. J. Parasitol. 36(14):1427-41; Efferth, et al. (2002) J. Mol. Med. 80(4):233-42; Jung and Schinazi (1994) Bioorg. Med. Chem. Lett. No. 7; 931-934; Kaptein, et al. (2006) Antiviral Res. 69(2):60-9; Paeshuyse, et al. (2006) Biochem. Biophys. Res. Commun. 15; 348(1):139-44; Razavi, et al. (2007) Int. J. Toxicol. 26(4):373-80; Li, et al. (2006) Int. Immunopharmacol. 6(8):1243-50; Wang, et al. (2006) Antimicrob. Agents Chemother. 50(7):2420-7; Xu, et al. (2007) Rheumatology (Oxford)).
Iron chelators are small molecules that bind to iron metal ions. Iron is critical for proliferation of cells and vital cellular processes, such as oxygen transport, energy production and DNA synthesis, which are dependent on iron-containing proteins and enzymes. Therefore, iron chelators are expected to possess various biological activities. It has been demonstrated that iron chelators have anti-tumor activities. Iron chelators induce cytotoxic effects on tumors by starving them of iron or by inducing oxidative stress in the tumors through redox perturbations. A number of iron chelators have been tested for anti-tumor activity in microbiology studies, animal models and human clinical trials (see, e.g., Lee, et al. (2006) J. Oral Pathol. Med. 35(4):218-26; Hoke, et al. (2005) Free Radic. Biol. Med. 1; 39(3):403-11; Shen, et al. (2005) In Vivo. 2005 19(1):233-6; Buss, et al. (2004) Curr. Top. Med. Chem. 4(15):1623-35; Buss, et al. (2003) Curr. Med. Chem.10(12):1021-34; Lovejoy and Richardson (2003) Curr. Med. Chem. 10(12):1035-49; Richardson (2002) Crit. Rev. Oncol. Hematol. 42(3):267-81).
The hydrazones constitute a class of iron-binding organic compounds, and certain members of the hydrazone class have been shown to inhibit cellular proliferation by removing iron from the active site of key enzymes, such as ribonucleoside reductase. In general, rapidly proliferating cancer cells are more sensitive to the hydrazones than the corresponding normal cells (see, e.g., Lovejoy, et al. (2006) Hemoglobin. 30(1):93-104; Walcourt, et al. (2004) Int. J. Biochem. Cell. Biol. 36(3):401-7; Lovejoy and Richardson. (2003) Curr. Med. Chem. 10(12):1035-49; Becker, et al. (2003) Br. J. Pharmacol. 138(5):819-30; Chaston, et al. (2003) Clin. Cancer. Res. 9(1):402-14; Lovejoy and Richardson (2002) Blood 100(2):666-76).
Attempts to combine artemisinin derivatives and iron chelators have been previously described in the literature to treat malaria, but have largely been therapeutically unsuccessful. For example, the synthesis of covalent conjugates between o-phenanthroline, a strong iron chelator, and an artemisinin-related endoperoxide for malaria therapy was reported in 1995 (Posner, et al. (1995) J. Med. Chem. 38(4):607-12), but the studied conjugates and related compounds were not particularly active antimalarial agents in vitro. Another group reported the synthesis of a series of covalent conjugates between artemisinin and a variety of iron chelators, including hydroxamates and phenolates, for malaria therapy (Yuthavong et al. (1995) J Med. Chem. 38(13):2311-6). Again, these conjugates did not demonstrate enhanced antimalarial activities, compared to artemisinin alone. These studies suggest that the simple conjugation of an iron chelator to artemisinin does not necessarily produce more active cytotoxic compounds. More recently, another group reported the synthesis of artemisinin conjugates with naphthol, an iron-binding molecule, for malaria therapy (Wang, et al. (1999) J. Chem. Soc. Perkin. Trans. 1827-1832). Based on the antimalarial activities of two stereoisomers, it was suggested that the naphthol group might assist iron in binding to the endoperoxide group before the artemisinin moiety is activated. To date none of the artemisinin-iron chelator compounds described above has been tested for activity in cancer or non-malarial proliferative diseases in published literature.
U.S. Pat. No. 5,225,427 discloses 10-substituted ether derivatives of dihydroartemisinin alleged to exhibit antimalarial and antiprotozoal activity.
U.S. Pat. No. 5,578,637 discloses methods of killing cancer cells wherein compounds having an endoperoxide moiety that is reactive with heme are administered under conditions which enhance intracellular iron concentrations. Endoperoxide bearing sesquiterpene including artemisinin and its analogs are preferred compounds.
U.S. Patent Application No. 2004/0058981 discloses methods for preventing or delaying the development of cancer by administering free radical-generating agents to a subject. Preferred compounds include endoperoxide bearing sesquiterpene compounds such as artemisinin and its analogs. Intracellular iron concentrations may be enhanced by the administration of iron salts or complexes.
U.S. Patent Application No. 2004/0067875 discloses covalent conjugates between artemisinin-related endoperoxides and iron-carrying proteins, such as holotransferrin, to treat cancer and infections by pathogens that bind iron-carrying proteins.
U.S. Patent Application No. 2006/0193778 and U.S. Pat. No. 6,743,893 disclose peptides discovered by phage display techniques that are capable of binding to and internalizing with the human transferring receptor, including the peptides HAIYPRH (SEQ ID NO: 1) and THRPPMWSPVWP (SEQ ID NO: 2).
U.S. Patent Application No. 2006/0142377 discloses orally active artemisinin-derived trioxane dimers suitable as orally active compounds, which demonstrate antimalarial and anti-tumor activities.
U.S. Patent Application No. 2007/0231300 discloses covalent conjugates between artemisinin-related endoperoxides and small peptides and organic compounds that bind to molecular cavities on the transferrin or lactoferrin receptor, and the use of these conjugates to treat cancer, hyperproliferative disorders, inflammatory diseases, and infections.
There is a need for artemisinin compounds having increased selectivity and efficacy for the treatment of proliferative cellular disorders, such as cancer, infections, and other hyperproliferative conditions dependent on iron for growth and virulence. The present invention seeks to fulfill these needs and provides further related advantages.